The FOXO-Forkhead transcription factors have important roles in regulation of the cell cycle, apoptosis, and stress responses in mammalian cells; however, little is known about their roles in tumorigenesis. We recently found that (1) nuclear FOXOSa inhibits cell proliferation and tumorigenesis of breast cancer cells; (2) phosphorylation of FOXOSa by kB kinase-p (IKK(3) leads to nuclear exclusion of FOXOSa, resulting in increases in cell proliferation and tumorigenesis; and (3) IKKp-mediated phosphorylation of FOXOSaon Ser644 leads to ubiquitination and proteasomal degradation of FOXOSa in the cytoplasm. Thus, IKKp is a key regulator of FOXOSafunction! However, regulation of the nucleocytoplasmic shuttling of FOXOSa is not fully understood, and the mechanisms by which FOXOSa promotes apoptosis and tumor suppression are largely unknown. The novel observations that IKKa and IKKy function in the nucleus to regulate NF-icB activity and that IKKp regulates nuclear extrusion of FOXOSa lead us to hypothesize that IKKa, IKKp, and IKKy may have a role in regulating the nucleocytoplasmic shuttling of FOXOSa and its activity in the nucleus, where it regulates the expression of growth-related proteins to elicit its tumor suppressive function. We also recently found that FOXOSa is associated with the p-transducing repeat-containing protein (p-TRCP) in vivo, suggesting that p-TRCP is a candidate E3 ubiquitin ligase that mediates the ubiquitination and proteasomal degradation of FOXOSa.Thus, we propose to study the mechanisms underlying the regulation ofFOXOSa activity and its role in promoting tumor suppression and to identify novel compounds that activate FOXO activity. The Specific Aims are to (1) investigate the roles of IKKa, IKKp, and IKKy in the regulation of nuclear FOXOSa activity and the role of p-TRCP ubiquitin ligase in proteolysis of FOXOSathrough the ubiquitination- dependent proteasome pathway in tumorigenesis; (2) identify molecular targets of FOXOSa and the regulation of these molecular targets by FOXOSa; and (3) develop a chemical genetic screen for identifying novel small molecules or peptides that can increase the transactivating activity of FOXO factors in breast cancer cells and investigate the roles and effects of these new compounds or peptides in tumor suppression in cell culture and breast cancer animal models. Our goal is to translate our knowledge of FOXOSa-mediated tumor suppression into new anticancer drug discovery for therapeutic intervention.